Use of peptides as therapeutic agent for autoimmune diseases and bone diseases

ABSTRACT

The present invention relates to use of peptides as a therapeutic agent, wherein it has been confirmed that the peptides of the present invention significantly inhibit the activity of T cells and the differentiation of T helper 17 cells (Th17 cells), which are associated with autoimmune disease, reduce the secretion of inflammatory cytokine IL-6, and have remarkable effects of treating and improving arthritis in an animal model of arthritis. Therefore, the peptides may be used as an active ingredient in therapeutic agents for various autoimmune diseases such as bone disease, inflammatory disease or rheumatoid arthritis.

This application is a National Phase of International Application No.PCT/KR2018/010874 filed on 14 Sep. 2018, which is claims priority ofKorean Patent Application No. 10-2017-0118950 filed on Sep. 15, 2017;Korean Patent Application No. 10-2017-0118952 filed on Sep. 15, 2017;Korean Patent Application No. 10-2018-0110481 filed on Sep. 14, 2018;and Korean Patent Application No. 10-2018-0110485 filed on Sep. 14,2018, which is hereby incorporated herein by reference in its entiretyfor all purposes.

TECHNICAL FIELD

The present invention relates to use of peptides as a therapeutic agentfor bone disease and autoimmune disease and more particularly, to apeptide consisting of an amino acid sequence represented by Formula 1 ofthe present invention, and use of the peptide for treating bone diseaseincluding osteoporosis, inflammatory disease, or autoimmune diseasesincluding rheumatoid arthritis.

BACKGROUND ART

Bone tissue consists of extracellular substances such as collagen andglycoprotein, and various kinds of cells such as osteoblasts,osteoclasts, and osteocytes. Particularly, the mutual balance ofosteoblasts and osteoclasts is essential for the formation of a healthyskeletal system. In other words, bone metabolism and bone remodeling areimportant for balanced activity between the osteoblasts that form a bonematrix and the osteoclasts that resorb the bone to maintain thehomeostasis of the bone.

The bone tissue consists of extracellular substances such as collagenand glycoprotein, and various kinds of cells such as osteoblasts,osteoclasts, and osteocytes. Further, the bone tissue is a metabolicorgan where bone resorption by osteoclasts and new bone matrix formationand mineralization by osteoblasts repeatedly occur, and the boneformation by the activity of osteoblasts is greater than bone resorptionby the activity of osteoclasts. The bone remodeling is a process ofremoving old bone after growth and replacing the removed bone with newbone, and hormones such as parathyroid hormone (PTH), calcitonin, andestrogen, various growth factors secreted from the bone tissue such asinsulin-like growth factor I (IGFI), and cytokines such as tumornecrosis factor-α (TNF-α) regulate the activity balance of osteoblastsand osteoclasts and maintain homeostasis. When the balance of theseosteoblasts and osteoclasts is broken, diseases such as osteoporosis orarthritis are induced.

In particular, when the balance of osteoblasts and osteoclasts isbroken, excessive bone destruction is caused by the osteoclasts, leadingto diseases such as osteoporosis. The osteoclasts as cells specializedfor the resorption of bone during the bone metabolism are formed througha differentiation program from monocytes or macrophages as progenitorcells. Further, since the osteoclasts secrete various collagenases andproteases to cause the bone resorption while binding to bone throughavβ3 integrin and the like and preparing an acidic environment,suppression of these osteoclasts may be an effective method of treatingbone diseases.

In addition, autoimmune diseases cause abnormalities of a human immunesystem so that self-cells attack self-cells. The human immune systembasically recognizes microorganisms invaded to the human body andgeneration of cancer cells as external antigens and has a strong powerto attack and remove the recognized microorganisms and cancer cells, butdoes not attack its own cells due to self-tolerance. This is called aself-tolerance phenomenon of the human body. However, when theself-tolerance of the immune system is destroyed, the human bodyactivates autoreactive T cells in response to self-cells (orautoantigens) and generates autoantibodies to constantly destroyself-cells and cause inflammation and immune responses.

Cells that specifically respond to antigens in the immune system includeT cells and B cells. When the T cells meet a specific antigen presentedby an antigen presenting cell, the T cells respond according to theantigen. When the antigen presented by the antigen presenting cell isrecognized as ‘non-self’, an immune response to remove the antigen isshown, and when the antigen is recognized as ‘self’, tolerance in whichthe immune response is ignored is shown. When the T cells are activatedagainst antigens, most B cells are successively activated and B cellsare converted into plasma cells to produce antibodies that specificallyrespond to the recognized specific antigen. Therefore, when tolerance isbroken in the human body and autoimmunity occurs, the T cells recognizeand activate autoantigens abnormally, the B cells are activated toproduce autoantibodies that respond to autoantigens, and then an immuneresponse to attack self-cells occurs in the body.

Similarly, even in organ transplant patients, when the immune systemrecognizes the transplanted tissue as ‘non-self’ after organtransplantation, organ transplant rejection reaction occurs to attackand remove the transplanted organ. In order to suppress the organtransplant rejection reaction, various immunosuppressants have beenused, such as suppressing the activation of immune cells and inhibitingthe migration of immune cells to transplanted organs, but the continuoususe of immunosuppressants causes various side-effects.

Meanwhile, recently, it has been found that Th17 cells as a CD4⁺ T cellsystem play a key role in the inflammatory induction and progression ofautoimmune diseases, and the importance of these Th17 cells is furtherincreasing by finding that the IL-17 secreted from these cells isdirectly associated with autoimmune diseases.

In addition, the Th17 cells are known to induce RANKL and variousinflammatory cytokines, which are the major causes of bone destruction(Chabaud and Miossec, 2001; Connell and McInnes, 2006), to furtheractivate inflammation and joint destruction mechanisms. Therefore, sincethe Th17 cells are recognized as key pathogens in signaling processesrelated to autoimmune diseases including rheumatoid arthritis and bonediseases, the discovery of candidates that effectively inhibit Th17 celldifferentiation has been required.

In addition, interleukin-6 (IL-6) is a cytokine involved in metabolism,regeneration and neural processes as well as inflammatory and infectiousresponses. Recently, the IL-6 has been mainly studied in autoimmunediseases such as rheumatoid arthritis and Crohn's disease, and it hasbeen known that the IL-6 is involved in differentiation of the Th17cells to act on the balance of Th17/Treg cells.

In addition, since the IL-6 is known to attract a bone marrow macrophageas an osteoclast precursor to a site of inflammation, stimulatedifferentiation into osteoclasts, activate the differentiatedosteoclasts, and resorb marginal bone, the IL-6 is recognized as a majortarget for the development of therapeutic agents for autoimmune diseasesand bone diseases.

Further, rheumatoid arthritis is an inflammatory disease characterizedby polyarthritis, and an autoimmune phenomenon is known as a mainmechanism. In the symptom, while inflammation of the articular synovialmembrane tissue occurs, macrophages, dendritic cells, T lymphocytes, Blymphocytes, and the like migrate to the synovial membrane tissue, andas a result, a joint fluid is increased and thus the joint is swollen tocause pain. As this inflammation continues, an inflammatory synovialmembrane tissue causes hyperplasia to destroy the bone and cartilage,thus a joint structure is modified and movement disorders occur. Inaddition, according to results of various studies, it has been knownthat in patients with rheumatoid arthritis, inflammatory cytokinesproduce collagenase and neutral protease in synovial membranefibroblasts and chondrocytes, and these produced enzymes destroycollagen and proteoglycans to destroy the articular cartilage.

Therefore, the present inventors have made efforts to develop a newtherapeutic agent for bone disease and autoimmune disease with aneffective therapeutic effect while minimizing side-effects as atherapeutic agent. As a result, the present inventors found that thepeptides prepared in the present invention may be usefully used for thetreatment of bone disease including osteoporosis, inflammatory disease,or autoimmune disease including rheumatoid arthritis and completed thepresent invention.

SUMMARY OF INVENTION Technical Problem

An object of the present invention is to provide peptides for preventingor treating bone disease, inflammatory disease, and autoimmune disease.

Solution to Problem

In order to achieve the object, the present invention provides a peptideconsisting of an amino acid sequence of Formula 1 represented by SEQ IDNO: 1 below, and a pharmaceutical composition for preventing andtreating bone disease, inflammatory disease or autoimmune disease orhealth foods for preventing and improving bone disease, inflammatorydisease or autoimmune disease, containing the peptide as an activeingredient.(X₁—X₂—X₃)_(n)  [Formula 1]

wherein, X₁ is any one selected from the group consisting of arginine(R), histidine (H), and lysine (K),

X₂ is aspartic acid (D) or glutamic acid (E),

X₃ is any one selected from the group consisting of glycine (G), alanine(A), valine (V), methionine (M), isoleucine (I) and leucine (L),

n is an integer from 1 to 10, and

the case where the amino acid sequence of Formula 1 above includes anRDG represented by SEQ ID NO: 2 and n=1 or 2 is excluded.

Advantageous Effects of Invention

It has been confirmed that the peptides of the present inventionsignificantly inhibit the activity of T cells and the differentiation ofT helper 17 cells (Th17 cells), which are associated with autoimmunedisease, significantly inhibit the secretion of IL-6, and have effectsof treating and improving arthritis in an arthritis animal model.Therefore, the peptides may be used as an active ingredient intherapeutic agents for bone disease, inflammatory disease or variousautoimmune diseases such as rheumatoid arthritis.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a diagram showing an active T cell population (%) by asynthetic peptide of the present invention.

FIG. 2 is a diagram showing a T cell activation inhibition rate (%) by asynthetic peptide of the present invention.

FIG. 3 is a diagram showing a Th17 cell differentiation inhibitoryeffect by a synthetic peptide of the present invention.

FIG. 4 is a diagram confirming an inflammatory cytokine IL-6 reductioneffect by a synthetic peptide of the present invention.

FIG. 5 is a schematic diagram schematically showing a process ofpreparing a collagen-induced arthritis mouse model and a time ofadministration of peptides according to the present invention.

FIG. 6 is a diagram showing an arthritis improvement effect of a peptidePep1 of the present invention.

FIG. 7 is a diagram showing an arthritis improvement effect of a peptidePep4 of the present invention.

FIG. 8 is a diagram showing an arthritis improvement effect of a peptidePep6 of the present invention.

FIG. 9 is a diagram showing an arthritis improvement effect of a peptidePep7 of the present invention.

FIG. 10 is a diagram showing an arthritis improvement effect of apeptide Pep8 of the present invention.

FIG. 11 is a diagram comparing arthritis improvement effects of peptidesof the present invention.

DESCRIPTION OF EMBODIMENTS

Hereinafter, terms of the present invention will be defined as follows.

In the present invention, general one-letter or three-letter codes fornaturally existing amino acids are used, and three-letter codesgenerally allowed for other amino acids, such as α-aminoisobutyric acid(Aib) and N-methylglycine (Sar) are also used. The amino acids mentionedherein as abbreviations are described according to the IUPAC-IUBnomenclature.

The “peptide” of the present invention refers to a polymer consisting oftwo or more amino acids linked by an amide bond (or peptide bond), andfor the purposes of the present invention, refers to a peptide having atherapeutic effect on bone disease, inflammatory disease, and autoimmunedisease.

The “stability” of the present invention means not only in-vivostability that protects the peptides of the present invention from theattack of protein cleavage enzymes in vivo, but also storage stability(e.g., room-temperature storage stability).

The “prevention” of the present invention means all actions that inhibitdisease or delay the onset of the disease by administration of apharmaceutical composition according to the present invention.

The “treatment” of the present invention means all actions that improveor advantageously change symptoms of the disease by the administrationof the pharmaceutical composition according to the present invention.

The “subject” of the present invention refers to a subject in need oftreatment for diseases, and more particularly, refers to mammals such ashuman or non-human primates, mice, dogs, cats, horses and cattle.

The “improvement” of the present invention means all actions that atleast reduce parameters associated with conditions to be treated, e.g.,the degree of symptoms.

Hereinafter, the present invention will be described in more detail.

The present invention provides a peptide consisting of an amino acidsequence of Formula 1 represented by SEQ ID NO: 1 below and apharmaceutical composition for preventing and treating bone disease,inflammatory disease, or autoimmune disease containing the peptide as anactive ingredient:(X₁—X₂—X₃)_(n)  [Formula 1]

wherein, X₁ is any one selected from the group consisting of arginine(R), histidine (H), and lysine (K) which are positive charged aminoacids, X₂ is aspartic acid (D) or glutamic acid (E) which are negativecharged amino acids, X₃ is any one selected from the group consisting ofglycine (G), alanine (A), valine (V), methionine (M), isoleucine (I) andleucine (L), n is an integer from 1 to 10, and the case where the aminoacid sequence of Formula 1 above includes an RDG represented by SEQ IDNO: 2 and n=1 or 2 is excluded.

The peptide may be prepared as various peptides using Formula 1 above,which are all included in the present invention. In addition, the X₁ ispreferably arginine (R) or histidine (H) and the X₃ is preferablyglycine (G) or alanine (A), but the present invention is not limitedthereto.

In addition, the n is preferably an integer of 1 to 6, and morepreferably an integer of 1 to 3.

The peptide of the present invention may be obtained by various methodswell-known in the art. As an example, the peptide may be prepared byusing polynucleotide recombination and a protein expression system orprepared by in-vitro synthesis through chemical synthesis such aspeptide synthesis, cell-free protein synthesis, and the like.

In addition, in order to obtain better chemical stability, enhancedpharmacological properties (half-life, absorbency, titer, efficacy,etc.), modified specificity (e.g., a wide biological activity spectrum),and reduced antigenicity, a protective group may bind to an N- orC-terminus of the peptide. Preferably, the protective group may be anacetyl group, a fluorenylmethoxy carbonyl group, a formyl group, apalmitoyl group, a myristyl group, a stearyl group, or polyethyleneglycol (PEG), but may include any ingredient that may enhance themodification of the peptide, particularly the stability of the peptide,without limitation.

The bone disease is preferably at least one selected from the groupconsisting of arthritis, osteoporosis, bone metastatic cancer, solidcancer bone metastasis, musculoskeletal complications due to solidcancer bone metastasis, hypercalcemia caused by malignant tumor,multiple myeloma, primary bone tumor, periodontal disease, inflammatoryalveolar bone resorption disease, inflammatory bone resorption disease,and Paget's disease, but is not limited thereto.

The inflammatory disease is preferably selected from the groupconsisting of atopy, psoriasis, dermatitis, allergies, arthritis,rhinitis, otitis media, laryngopharyngitis, tonsillitis, cystitis,nephritis, pelvic inflammatory, Crohn's disease, ulcerative colitis,ankylosing spondylitis, systemic lupus erythematodes (SLE), asthma,edema, delayed allergy (Type IV allergy), transplant rejection,graft-versus-host disease, autoimmune encephalomyelitis, multiplesclerosis, inflammatory bowel disease, cystic fibrosis, diabeticretinopathy, ischemic-reperfusion injury, vascular restenosis,glomerulonephritis, and gastrointestinal allergy, but is not limitedthereto.

The autoimmune disease is preferably selected from the group consistingof rheumatoid arthritis, Sjogren's syndrome, systemic sclerosis,polymyositis, systemic angitis, mixed connective tissue disease, Crohn'sdisease, Hashimoto's disease, Grave's disease, Goodpasture's syndrome,Guillain-Barre syndrome, idiopathic thrombocytopenic purpura, irritablebowel syndrome, myasthenia gravis, narcolepsy, vulgaris ulcer,pernicious anemia, primary biliary cirrhosis, ulcerative colitis,vasculitis, Wegener's granulomatosis, and psoriasis, but is not limitedthereto.

In addition, since the same therapeutic effect may be exhibited even byusing polynucleotides encoding the peptide of the present invention, itis obvious that the polynucleotides encoding the peptide of the presentinvention are also included in the present invention.

In a specific embodiment of the present invention, the present inventorsprepared various peptides using Formula 1 above [(X₁—X₂—X₃)_(n)] (seeTable 1).

In addition, the present inventors confirmed a T cell activityinhibitory effect of the peptides, and as a result, the peptides ofTable 1 significantly inhibit the T cell activity by an average of 8%,and the T cell activity inhibitory effect of peptides randomly selectedamong the peptides was shown in FIGS. 1 and 2 (see FIGS. 1 and 2 ).

In addition, the present inventors confirmed that a synthetic peptideprepared in Example 1 inhibits Th17 cell differentiation, and as aresult, confirmed that the peptides of the present inventionsignificantly inhibit the Th17 cell differentiation (see FIG. 3 ).

In addition, the present inventors confirmed an IL-6 secretioninhibitory effect of the peptides, and as a result, confirmed that thepeptides of the present invention significantly inhibit the secretion ofIL-6 similarly to a positive control group (see FIG. 4 ).

Further, the present inventors prepared a rheumatoid arthritis animalmodel (see FIG. 5 ) and then confirmed a therapeutic effect of thepeptides of the present invention, and as a result, confirmed that thepeptides showed a significant arthritis improvement effect,particularly, a similar effect compared to a positive control group MTX(see FIGS. 6 to 11 ).

Therefore, since it has been confirmed that the peptides of the presentinvention significantly inhibit the activity of T cells and thedifferentiation of T helper 17 cells (Th17 cells), which are associatedwith autoimmune diseases, and inhibit the secretion of IL-6 similarly tothe positive control group to have remarkable effects of treating andimproving arthritis in an arthritis animal model, the peptides may beused as an active ingredient in therapeutic agents for bone disease,inflammatory disease or various autoimmune diseases such as rheumatoidarthritis.

On the other hand, the peptides of the present invention orpolynucleotides encoding the same may be carried in pharmaceuticallyacceptable carriers such as colloidal suspensions, powders, saline,lipids, liposomes, microspheres, or nano spherical particles. Thesepeptides or polynucleotides may form a complex with a vehicle or beassociated with the vehicle and may be carried in vivo by using vehiclesystems known in the art, such as lipids, liposomes, microparticles,gold, nanoparticles, polymers, condensation reagents, polysaccharides,polyamino acids, dendrimers, saponins, adsorption enhancing substancesor fatty acids.

Besides, the pharmaceutically acceptable carrier may include lactose,dextrose, sucrose, sorbitol, mannitol, starch, acacia, rubber, calciumphosphate, alginate, gelatin, calcium silicate, microcrystallinecellulose, polyvinylpyrrolidone, cellulose, water, syrup,methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc,magnesium stearate, and mineral oil, which are generally used inpreparation, but is not limited thereto. Further, the pharmaceuticalcomposition may further include a lubricant, a wetting agent, asweetening agent, a flavoring agent, an emulsifying agent, a suspendingagent, a preservative, and the like, in addition to the ingredients.

The pharmaceutical composition of the present invention may beadministered orally or parenterally (e.g., applied intramuscularly,intravenously, intraperitoneally, subcutaneously, intradermally, ortopically) according to a desired method, and a dose thereof variesdepending on the condition and weight of a patient, a degree of disease,a drug form, and route and time of administration, but may beappropriately selected by those skilled in the art.

The pharmaceutical composition of the present invention is administeredin a pharmaceutically effective dose. In the present invention, the“pharmaceutically effective dose” refers to an amount sufficient totreat the diseases at a reasonable benefit/risk ratio applicable tomedical treatment, and an effective dose level may be determinedaccording to elements including the type and severity of disease of apatient, activity of a drug, sensitivity to a drug, a time ofadministration, a route of administration and an emission rate, durationof treatment, and simultaneously used drugs, and other elementswell-known in the medical field. The pharmaceutical compositionaccording to the present invention may be administered as an individualtherapeutic agent or in combination with other therapeutic agents forbone disease, inflammatory disease, or autoimmune disease, andadministered simultaneously, separately, or sequentially withconventional therapeutic agents for bone disease, inflammatory disease,or autoimmune disease, and may be administered singly or multiply. It isimportant to administer an amount capable of obtaining a maximum effectwith a minimal amount without side-effects by considering all theelements, and this may be easily determined by those skilled in the art.

Specifically, the effective dose of the pharmaceutical composition ofthe present invention may vary depending on the age, sex, condition, andweight of a patient, absorbance of an active ingredient in vivo, aninactivation rate, an excretion rate, a disease type, and drugs to beused in combination, and may be increased or decreased according to aroute of administration, the severity of obesity, sex, weight, age, andthe like.

Further, the present invention provides health foods for preventing andimproving bone disease, inflammatory disease or autoimmune diseasecontaining the peptide of the present invention or polynucleotidesencoding the same as an active ingredient.

The health foods may be used simultaneously or separately with a drugfor treatment before or after the onset of the corresponding disease inorder to prevent or improve the disease.

In the health foods of the present invention, the active ingredient maybe added to the foods as it is or used with other foods or foodingredients, and may be appropriately used according to a generalmethod. The mixing amount of the active ingredients may be suitablydetermined according to the purpose of use thereof (prevention orimprovement). In general, in preparation of foods or beverages, thecomposition of the present invention may be added preferably in anamount of 15 wt % or less, more preferably 10 wt % or less with respectto raw materials. However, in the case of long-term ingestion for thepurpose of health and hygiene or health regulation, the amount may beused below the above range.

The health foods of the present invention may contain other ingredientsas essential ingredients without particular limitation, in addition tothe active ingredients. For example, like general beverages, variousflavoring agents or natural carbohydrates may be contained as anadditional ingredient. Examples of the above-mentioned naturalcarbohydrates may include monosaccharides, such as glucose, fructose,and the like; disaccharides, such as maltose, sucrose, and the like; andgeneral sugars, such as polysaccharides such as dextrin, cyclodextrin,and the like, and sugar alcohols such as xylitol, sorbitol, erythritol.In addition to those described above, as the flavoring agent, naturalflavoring agents (thaumatin, stevia extracts (e.g., rebaudioside A,glycyrrhizin, etc.)) and synthetic flavoring agents (saccharin,aspartame, etc.) may be advantageously used. The ratio of the naturalcarbohydrates may be appropriately determined by the selection of thoseskilled in the art.

In addition, the health foods of the present invention may containvarious nutrients, vitamins, minerals (electrolytes), flavoring agentssuch as synthetic and natural flavoring agents, coloring agents andenhancers (cheese, chocolate, etc.), pectic acid and salts thereof,alginic acid and salts thereof, organic acid, a protective colloidalthickener, a pH adjusting agent, a stabilizer, a preservative, glycerin,alcohol, a carbonic acid agent used in a carbonated drink, and the like.These ingredients may be used independently or in combination, and theratio of these additives may also be appropriately selected by thoseskilled in the art.

Hereinafter, the present invention will be described in detail byExamples and Test Examples.

However, the following Examples and Test Examples are just illustrativeof the present invention, and the contents of the present invention arenot limited to the following Examples and Test Examples.

<Example 1> Preparation of Peptides

Various peptides were prepared based on the following Formula 1.Subsequently, each of the synthesized peptides was purified andseparated using high performance liquid chromatography (SHIMADZUProminence HPLC), and a column used is a Shiseido capcell pak C18 column(4.6×50 mm). In addition, the mass of each synthesized peptide wasconfirmed by using a mass spectrometer (HP 1100 series LC/MSD).(X₁—X₂—X₃)_(n)  [Formula 1]

wherein, X₁ is any one selected from the group consisting of arginine(R), histidine (H), and lysine (K),

X₂ is aspartic acid (D) or glutamic acid (E),

X₃ is any one selected from the group consisting of glycine (G), alanine(A), valine (V), methionine (M), isoleucine (I) and leucine (L),

n is an integer from 1 to 10, and

the case where the amino acid sequence of Formula 1 above includes anRDG represented by SEQ ID NO: 2 and n=1 or 2 is excluded.

In addition, the peptides synthesized by the method were listed in Table1 below.

TABLE 1 Synthetic Synthetic No. peptide No. peptide  1 RDA 44 REGREG  2RDV 45 REGREGREG  3 RDM 46 REGREGREGREG  4 RDI 47 REVREV  5 RDL 48REVREVREV  6 REG 49 REVREVREVREV  7 REA 50 HDGHDG  8 REV 51 HDGHDGHDG  9REM 52 HDGHDGHDGHDG 10 REI 53 HDMHDM 11 REL 54 HDMHDMHDM 12 HDG 55HDMHDMHDMHDM 13 HDA 56 HEGHEG 14 HDV 57 HEGHEGHEG 15 HDM 58 HEGHEGHEGHEG16 HDI 59 KDGKDG 17 HDL 60 KDGKDGKDG 17 HEG KDGKDGKDGKDG 18 HEA 61KDGKDGKDGKDGKDG 19 HEV 62 KEGKEG 20 HEM 63 KEGKEGKEG 21 HEI 64KEGKEGKEGKEG 22 HEL 65 KEGKEGKEGKEGKEG 23 KDG 66 KEAKEA 24 KDA 67KEAKEAKEA 25 KDV 68 KEAKEAKEAKEA 26 KDM 69 KEAKEAKEAKEAKEA 27 KDI 70KDAKDA 28 KDL 71 KDAKDAKDAKDA 29 KEG 72 KDAKDAKDAKDAKDA 30 KEA 73KDAKDAKDAKDAKDAKDA 31 KEV 74 KEVKEV 32 KEM 75 KEVKEVKEV 33 KEI 76KEVKEVKEVKEV 34 KEL 77 KEVKEVKEVKEVKEV 35 RDARDA 78 HEMHEM 36 RDARDARDA79 HEMHEMHEM 37 RDARDARDARDA 80 HEMHEMHEMHEM 38 RDVRDV 81HEMHEMHEMHEMHEM 39 RDVRDVRDV 82 REMREM 40 RDVRDVRDVRDV 83 REMREMREM 41RDMRDM 84 REMREMREMREM 42 RDMRDMRDM 85 REMREMREMREMREM 43 RDMRDMRDMRDM86 REMREMREMREMREMREM

<Test Example 1> Confirmation of T Cell Activity Inhibitory Effect

In order to confirm the T cell activity inhibitory effect of thesynthetic peptide prepared in Example 1, an ex vivo activity inhibitiontest was performed using T cells extracted from the lymph nodes of amouse.

Specifically, at first, in order to induce activation of T cells, CD3antibodies were coated on a 96 well plate and incubated overnight at 4°C. to prepare 96 wells attached with the CD3 antibodies. Thereafter,naive T cells extracted from the mouse were seeded in the 96 well plateby 1×10⁵/well, treated with each of the synthetic peptides prepared inExample 1 and incubated for 18 hours, and then the population of theactive T cells was confirmed by flow cytometry. To this end, the samenumber of cells were collected from each incubated group and washed withPBS, and then the collected cells were subjected to staining by usingrabbit anti-mouse CD4 as a helper T cell marker and a rabbit anti-mouseCD69 antibody as a T-cell activation marker. The cells were washed withPBS and then CD4+CD69+ T cell population was analyzed.

As a result, it was confirmed that the peptides synthesized in Example 1significantly inhibited T cell activity by an average of 8%.

In addition, among the peptides synthesized in Example 1, the T cellactivity inhibitory effect of the peptides shown in Table 2 was shown inFIGS. 1 and 2 .

Specifically, it was confirmed that compared to a group that did notinduce activation (1.6%), active T cells increased by 85.2% in a groupactivated with the CD3 antibody, and decreased by 77% to 82% in a grouptreated with each synthetic peptide. In addition, it was confirmed thatthe active T cell inhibition rate of each synthetic peptide was about8%, similarly to the peptides synthesized in Example 1.

Therefore, it was confirmed that the synthetic peptides of the presentinvention may be used to treat autoimmune diseases by significantlyinhibiting the T cell activity (FIGS. 1 and 2 ).

TABLE 2 Name of Amino acid peptide sequence Pep1 KDGKDG Pep2 KEGKEG Pep3KEAKEA Pep4 KDAKDA Pep5 KDGKDGKDG Pep6 KEGKEGKEG Pep7 KEAKEAKEA Pep8KDAKDAKDA

<Test Example 2> Confirmation of Th 17 Cell Differentiation InhibitoryEffect

In order to confirm the efficacy of the synthetic peptides prepared inExample 1 to inhibit Th17 cell differentiation, naive CD4⁺ T cellsextracted from lymph nodes of the mouse were treated with IL-6 20 ng/mland TGF-beta 5 ng/ml together with TCR activation to inducedifferentiation into Th17 cells.

At the same time, three peptides Pep1, Pep6, and Pep8 disclosed in Table2 of Test Example 1 were treated at a concentration of 10 ng/ml to 1000ng/ml, respectively. Then, after incubation for 3 days, CD4+IL-17+ Tcell population was analyzed.

As a result, as shown in FIG. 3 , there was a tendency of about 2.5-foldincrease in a group that induced Th17 differentiation (3.14%), comparedto a group that did not induce differentiation into Th17 cells (1.2%),and in a group treated with peptides Pep1, Pep6, and Pep8, it wasconfirmed that a ratio of Th17 cells was reduced similarly to the groupthat did not induce differentiation (FIG. 3 ).

<Test Example 3> Confirmation of Inflammatory Cytokine IL-6 ReductionEffect

In order to confirm the efficacy of the synthetic peptides prepared inExample 1 to inhibit inflammatory cytokine IL-6 secretion, a THP-1monocytic cell line was treated with PMA 50 ng/ml for 48 hours to bedifferentiated into M1 macrophages. After treatment with LPS 10 ng/mland IFN-gamma 20 ng/ml to the differentiated M1 macrophages, a positivecontrol group MTX or the synthetic peptides of the present inventionwere co-treated at a concentration of 100 ng/ml, respectively.Thereafter, after incubation for 24 hours, IL-6 ELISA was performedusing a cultured sup.

As a result, as shown in FIG. 4 , it was confirmed that IL-6 wassignificantly reduced by peptides Pep2, Pep3, Pep5, Pep7, and Pep8 ofthe present invention, similarly to the positive control group (FIG. 4).

<Test Example 4> Confirmation of Rheumatoid Arthritis Treatment EffectUsing Collagen-Induced Arthritis (CIA) Mouse Model

<4-1> Preparation of Rheumatoid Arthritis Mouse Model

In order to confirm a rheumatoid arthritis improvement effect of thepeptides prepared in Example 1, a rheumatoid arthritis mouse model wasprepared with reference to a known literature (Nat Protoc. 2007; 2 (5):1269-75.).

Specifically, a CIA mouse model is a mouse model most commonly used in arheumatoid arthritis animal test as an autoimmune disease arthritismodel having characteristics similar to human rheumatoid arthritis. Inthe CIA mouse model, bovine type II collagen (Chondrex, USA) was mixedand emulsified with a Freund's complete adjuvant (Chondrex, USA) at 1:1,and then first immunization was performed by injecting 50 μl of theemulsified collagen solution intradermally into a 6-week-old DBA/1Jmouse tail. On 2 weeks after the first immunization, bovine type IIcollagen was mixed and emulsified with a Freund's incomplete adjuvant(Chondrex, USA) at 1:1, and then second immunization (boosting) wasperformed by injecting 50 μl of the emulsified collagen solutioninduced) intradermally into the mouse tail. After the secondimmunization, each peptide was intraperitoneally administered 3 times aweek from the following day to observe a therapeutic effect of thepeptides for rheumatoid arthritis. The administered peptides wereselected as five peptides Pep1, Pep4, Pep6, Pep7, and Pep8 shown inTable 2 (FIG. 5 ).

<4-2> Confirmation of Treatment Effect Using Rheumatoid Arthritis MouseModel

In order to examine the progression of rheumatoid arthritis according tothe peptide treatment of the present invention, the severity ofrheumatoid arthritis over time was evaluated and measured by arheumatoid arthritis progress index.

Two observers, who did not know specific test conditions, evaluated theprogression of arthritis three times a week. At this time, the arthritisprogress index was evaluated as Points 0 to 4 per leg in accordance withthe arthritis progress evaluation criteria by Rossoliniec, etc. in Table2 below and shown to total Points 0 to 16 (sum of four legs).Thereafter, an average value of the results evaluated by the twoobservers was calculated to quantify the severity of arthritis.

TABLE 3 Score Symptoms Point 0 There was no edema or swelling Point 1Mild edema and redness confined to the foot or ankle joint wereobserved. Point 2 Mild swelling and redness across the tarsal bone atthe ankle joint were observed. Point 3 Moderate swelling and rednessacross the tarsal bone at the ankle joint were observed. Point 4 Therewere edema and redness throughout the leg from the ankle, and jointstiffness was observed.

As a result, as shown in FIGS. 6 to 11 , it can be seen that arthritisscores are significantly increased in CIA-induced mice (Vehicle control;PBS) as compared to a normal mouse group, and it was confirmed that theefficacy of arthritis improvement was shown in a group intraperitoneallyadministered with five peptides Pep1, Pep4, Pep6, Pep7, and Pep8,respectively. In addition, it was confirmed that the similar efficacywas shown compared to the positive control group MTX (FIGS. 6 to 11 ).

Therefore, it was confirmed that the peptides of the present inventionmay be used as therapeutic agents for various bone diseases includingarthritis and autoimmune diseases.

The invention claimed is:
 1. A synthetic peptide consisting of an aminoacid sequence of Formula 1 represented by SEQ ID NO: 1 below:(X₁—X₂—X₃)_(n)  [Formula 1] wherein, X₁ is lysine (K), X₂ is asparticacid (D), X₃ is alanine (A), and n is an integer from 2 to
 10. 2. Thesynthetic peptide according to claim 1, wherein an N- or C-terminus ofthe peptide is bound to a protective group selected from the groupconsisting of an acetyl group, a fluorenylmethoxy carbonyl group, aformyl group, a palmitoyl group, a myristyl group, a stearyl group, andpolyethylene glycol (PEG).
 3. A method for treating rheumatoid arthritiscomprising administering a synthetic peptide consisting of an amino acidsequence of Formula 1 represented by SEQ ID NO: 1 below in apharmaceutically effective dose to a subject with rheumatoid arthritis:(X₁—X₂—X₃)_(n)  [Formula 1] wherein, X₁ is lysine (K), X₂ is asparticacid (D), X₃ is alanine (A), and, n is an integer from 2 to 10.